Chagas'disease is a tropical parasitic disease that develops over a number of years in individuals that are chronically infected with the protozoan parasite, Trypanosoma cruzi. As an obligate intracellular parasite, T. cruzi exploits host cell metabolic and cellular processes to fuel its own growth in the host cell cytoplasm. While intracellular amastigotes represent the critical stage in the T. cruzi life cycle to target for chemotherapeutic intervention in both acute and chronic Chagas'disease, very little is known regarding the host cell requirements to support intracellular growth and survival of this pathogen. Here we propose to conduct a genome-wide RNA interference screen to identify critical host cell regulators of T. cruzi infection of mammalian cells. The goal of this work is to obtain a global view of the critical biology at the T. cruzi-host cell interface by moving away from single gene targets, toward an integrated view of the host cell biological pathways and cellular networks required to support intracellular T. cruzi infection. The proposed siRNA screen will exploit a pre-existing cell-based high-throughput assay for T. cruzi growth, which measures 2-galactosidase activity associated with stable transgenic T. cruzi- 2-galactosidase-expressing parasites in a 384-well plate format. We will screen an arrayed Dharmacon siRNA library containing 19,470 siRNA pools targeting the majority of the human genome, to identify human genes (in HeLa cells) that are critical for supporting T. cruzi entry and intracellular growth. We will independently validate the top hits in secondary screening efforts and prioritize these based on biological network analysis using available bioinformatics tools such as Ingenuity Pathway Analysis" and GeneGo's MetaCore Analysis. In the second phase of the study, a focused image-based screen will be conducted to broadly categorize siRNA hits into those associated with pre-replication events from those that impact intracellular replication. Results from our screening efforts and the preliminary functional analysis will be made available to the broader Chagas'disease community and therefore provide an immediate opportunity to exploit our findings for mechanistic studies of critical aspects of Chagas'disease pathogenesis from the standpoint of integrated host cell networks. PUBLIC HEALTH RELEVANCE: Chagas'disease is a tropical parasitic disease that develops over a number of years in individuals that are chronically infected with the protozoan parasite, Trypanosoma cruzi. As a leading cause of heart failure in Latin America, Chagas'disease represents a public health problem of exceptional importance in endemic countries as well as an emerging immigrant health issue in the United States. T. cruzi is an obligate intracellular parasite that exploits host cell metabolic and cellular processes to fuel its growth in the host cell cytoplasm. Although intracellular amastigotes represent the critical stage in the T. cruzi life cycle to target for chemotherapeutic intervention in both acute and chronic Chagas'disease, very little is known regarding the host cell requirements to support intracellular growth and survival of this pathogen. The study proposed here seeks to identify host cell pathways, on a genome-wide scale, that are critical for supporting intracellular T. cruzi infection in host cells. Results from this work will provide immediate and important insights into T. cruzi pathogenesis and guide efforts toward effective prevention or control of Chagas'disease.